We propose to continue our studies of the effects of increased hydrostatic pressure (0-10 kbar) on proteins, membranes and their interactions with other molecules. Pressures of this magnitude do not distort covalent bonds. Rather, such pressures perturb hydrophobic, electrostatic and hydrogen bonding interactions, which are the dominant forces determining the structure and function of macromolecules. These non-covalent interactions each display a different sensitivity to pressure, or stated equivalently, characteristic volume changes. Hence, the effects of pressure on the stability and/or association reactions of macromolecules may be used to infer the nature of the molecular interactions responsible for stability or binding. Fluorescence methods will be utilized, with a new emphasis on the variable-frequency phase-modulation fluorometer which is currently under construction. The following phenomena will be studied: 1) The effects of pressure on protein folding and dynamics, as revealed by the penetration of water-soluble quenchers and the time-dependent decays of tryptophan anisotropies. The latter will be determined by frequency-domain measurements. 2) The effect of pressure on the heme-binding site of myoglobin, as revealed by geometric reorientation of aryl anthracenes, and by the pressure-dependent binding of a fluorescence probe. 3) The effect of pressure on the partitioning of fluorophores and a fluorescent anesthetic into micelles and membranes. And 4) The effects of pressure on lateral phase separations in model membranes. Since the effects of pressure on proteins are smaller than on membranes we propose to construct a second pressure cell, capable of pressures to 10 kbar.